Performance of a pilot-scale packed bed reactor for perchlorate reduction using a sulfur oxidizing bacterial consortium

A novel sulfur‐utilizing perchlorate reducing bacterial consortium successfully treated perchlorate (ClO) in prior batch and bench‐scale packed bed reactor (PBR) studies. This study examined the scale up of this process for treatment of water from a ClO and RDX contaminated aquifer in Cape Cod Massachusetts. A pilot‐scale upflow PBR (～250‐L) was constructed with elemental sulfur and crushed oyster shell packing media. The reactor was inoculated with sulfur oxidizing ClO reducing cultures enriched from a wastewater seed. Sodium sulfite provided a good method of dissolved oxygen removal in batch cultures, but was found to promote the growth of bacteria that carry out sulfur disproportionation and sulfate reduction, which inhibited ClO reduction in the pilot system. After terminating sulfite addition, the PBR successfully removed 96% of the influent ClO in the groundwater at an empty bed contact time (EBCT) of 12 h (effluent ClO of 4.2 µg L^?1). Simultaneous ClO and NO reduction was observed in the lower half of the reactor before reactions shifted to sulfur disproportionation and sulfate reduction. Analyses of water quality profiles were supported by molecular analysis, which showed distinct groupings of ClO and NO degrading organisms at the inlet of the PBR, while sulfur disproportionation was the primary biological process occurring in the top potion of the reactor. Biotechnol. Bioeng. 2012; 109:637–646. © 2011 Wiley Periodicals, Inc.     

Modelling of sulfur oxidation from elemental sulfur

An elemental S oxidation model has been developed which combines a maximum S release rate with modifiers for temperature and soil moisture conditions. This model has been combined with a pasture growth and CNSP nutrient cycling model to match S oxidation rate to pasture S demand. In two Southern Australian enviroments, 100m elemental S was superior to 200m particles whilst in Northern Australia the 200m particles were superior. These models can be used to match S release to plant demand.     

Effluent recirculation to improve perchlorate reduction in a fixed biofilm reactor

The effect of effluent recirculation on perchlorate reduction in a nominally plug-flow fixed biofilm reactor was studied in two cases: influent concentrations of 10 and 400 microg/L at low hydraulic loading rates (1.9 and 37.5 m(3)/m(2)/day without and with recirculation, respectively) and after a step increase in perchlorate concentration to 1,000 microg/L at the higher hydraulic loading rate (5 and 100 m(3)/m(2)/day without and with recirculation, respectively). Complete perchlorate reduction was sustained for influent concentrations of 400 and 10 microg/L in both flow regimes at the lower hydraulic loading rates. Reactor tracer profiles showed that biofilm diffusion had a more significant effect on mass transfer in the plug flow reactor compared with recirculation. The recirculation bioreactor acclimated more rapidly to increased hydraulic and perchlorate mass loading rates with significantly lower effluent perchlorate compared to the plug flow reactor: 16 microg/L versus 46 microg/L, respectively, although complete perchlorate removal was not achieved in either flow regime after 21 days acclimation to the higher loading. Total biofilm mass was more uniformly distributed in the recirculation reactor which may have contributed to better performance under increased perchlorate loading.     

Hydrogenotrophic denitrification and perchlorate reduction in ion exchange brines using membrane biofilm reactors

Halophilic (salt loving), hydrogenotrophic (H₂ oxidizing) denitrifying bacteria were investigated for treatment of nitrate <$>({\rm NO}_3^ ‐ )<$> and perchlorate <$>({\rm ClO}_4^ ‐ )<$> contaminated groundwater and ion exchange (IX) brines. Hydrogenotrophic denitrifying bacteria were enriched from a denitrifying wastewater seed under both halophilc and non‐halophilc conditions. The cultures were inoculated into bench‐scale membrane biofilm reactors (MBfRs) with an “outside in” configuration, with contaminated water supplied to the lumen of the membranes and H₂ supplied to the shell. Abiotic mass transfer tests showed that H₂ mass transfer coefficients were lower in brines than in tap water at highest Reynolds number, possibly due to increased transport of salts and decreased H₂ solubility at the membrane/liquid interface. An average <$>{\rm NO}_3^ ‐ <$> removal efficiency of 93% was observed for the MBfR operated in continuous flow mode with synthetic contaminated groundwater. Removal efficiencies of 30% for <$>{\rm NO}_3^ ‐ <$> and 42% for <$>{\rm ClO}_4^ ‐ <$> were observed for the MBfR operated with synthetic IX brine in batch operating mode with a reaction time of 53 h. Phylogenetic analysis focused on the active microbial community and revealed that halotolerant, <$>{\rm NO}_3^ ‐ <$> ‐reducing bacteria of the bacterial classes Gamma‐Proteobacteria and Sphingobacteria were the metabolically dominant members within the stabilized biofilm. This study shows that, despite decreased H₂ transfer under high salt conditions, hydrogenotrophic biological reduction may be successfully used for the treatment of <$>{\rm NO}_3^ ‐ <$> and <$>{\rm ClO}_4^ ‐ <$> in a MBfR. Biotechnol. Bioeng. 2009; 104: 483–491 © 2009 Wiley Periodicals, Inc.     

Removal of nitrate and hexavalent uranium from groundwater by sequential treatment in bioreactors packed with elemental sulfur and zero‐valent iron

The bioreduction of soluble hexavalent uranium (U^VI) to insoluble tetravalent uranium (U^IV) is an attractive bioremediation strategy for the clean‐up of contaminated groundwater. High levels of the common occurring co‐contaminant, nitrate ( ), can potentially interfere with uranium bioremediation. In this study, treatment of a synthetic groundwater containing a mixture of and U^VI was investigated in a sulfur–limestone autotrophic denitrifying (SLAD) bioreactor that was coupled in series with a bioreactor packed with zero‐valent iron (Fe⁰, ZVI) and sand. An additional aim of the study was to explore the possible role of biological activity in enhancing the reduction of U^VI by Fe⁰. The SLAD reactor removed efficiently (99.8%) at loadings of up to 20 mmol L d^?1, with near stoichiometric conversion to benign dinitrogen gas (N₂). The ZVI bioreactor subsequently removed uranium (99.8%) at high (0.22 mM) and low (0.02 mM) influent concentrations of the radionuclide. Aqueous uranium was reliably eliminated to below the maximum contaminant level of 30 µg L^?1 (0.13 µM) when the ZVI reactor was operated at average empty bed hydraulic retention times as low as 2.3 h, demonstrating the feasibility of the sequential treatment strategy in packed bed bioreactors. Sequential extraction of the ZVI reactor packing confirmed that uranium was immobilized as U^IV. Uranium removal was enhanced by microbial activity as confirmed by the increased rate of uranium removal in batch assays inoculated with effluent from the ZVI bioreactor and spiked with Fe⁰ compared to abiotic controls. Biotechnol. Bioeng. 2010;107: 933–942. © 2010 Wiley Periodicals, Inc.     

Bioremediation of Perchlorate-Contaminated Groundwater Using a Packed Bed Biological Reactor

The objective of this study was to assess the efficacy of a bench-scale, acetate-fed, packed bed bioreactor (PBR) to treat low concentrations (>1 mg L^?1) of perchlorate (ClO₄ ^?) in groundwater collected from an impacted site. The PBR consisted of a cylindrical plexiglass column packed with Celite, a diatomaceous earth product, as a solid support medium. The reactor was inoculated with a ClO_?4 ^?-reducing bacterial isolate, perclace. Results showed that with influent ClO₄ ^? concentrations of approximately 800 μg L^?1, nondetectable effluent concentrations (>4 μg L^?1) were achieved with the PBR/perclace system at residence time as low as 0.3 h. Influent acetate concentrations of less than 500 mg L^?1 yielded nondetectable effluent ClO^? ₄ concentrations, and acetate concentrations generally less than 50 mg L^?1 were present in the effluent. Nitrate (NO^? _?3) was also removed in this system, while sulfate (SO₄ ^2?) reduction was not observed. The pH remained relatively constant during the process.     

Chemolithotrophic growth of the phototrophic sulfur bacterium Thiocapsa roseopersicina

Chemotrophic growth capacities of the purple sulfur bacterium Thiocapsa roseopersicina strain M1 were studied in continuous culture under thiosulfate limitation.Pigment synthesis was completely inhibited upon a shift from anaerobic to semi-aerobic conditions (52 μM O₂) in the light, but no active breakdown occurred. During the transient state, the cells grew in a mixed photo- and chemolithotrophic mode; the specific respiration rate gradually increased with a concomitant drop in the bacteriochlorophyll a content. Photolithotrophically grown cells have the ability to respire. It was concluded that photosynthesis and respiration compete for electrons, but that photosynthesis is preferred under electron donor-limiting conditions, when the cells still contain large amounts of pigments. Eventually, a fully chemolithotrophic steady state was attained.The chemolithotropic growth of T. roseopersicina was studied in the dark under semiaerobic conditions at various dilution rates. The maximum specific growth rate was 68% of the maximum attainable growth rate under photolithotrophic conditions. The growth affinity for thiosulfate was high (K_m = 1.5 μM). The yield on thiosulfate under chemolithotrophic conditions exceeded that of thiobacilli. Oxygen uptake was studied in short-term experiments. It was shown that respiration in T. roseopersicina has a K_m of approx. 1 μM O₂. the ecological importance for T. roseopersicina of chemolithotrophic growth and pigment content is discussed with respect to the occurrence of T. roseopersicina in laminated microbial ecosystems and its possible competition with colorless sulfur bacteria.     

Growth of the facultative anaerobe Shewanella putrefaciens by elemental sulfur reduction

The growth of bacteria by dissimilatory elemental sulfur reduction is generally associated with obligate anaerobes and thermophiles in particular. Here we describe the sulfur-dependent growth of the facultatively anaerobic mesophile Shewanella putrefaciens. Six of nine representative S. putrefaciens isolates from a variety of environments proved able to grow by sulfur reduction, and strain MR-1 was chosen for further study. Growth was monitored in a minimal medium (usually with 0.05% Casamino Acids added as a growth stimulant) containing 30 mM lactate and limiting concentrations of elemental sulfur. When mechanisms were provided for the removal of the metabolic end product, H2S, measurable growth was obtained at sulfur concentrations of from 2 to 30 mM. Initial doubling times were ca. 1.5 h and substrate independent over the range of sulfur concentrations tested. In the cultures with the highest sulfur concentrations, cell numbers increased by greater than 400-fold after 48 h, reaching a maximum density of 6.8 x 10(8) cells ml-1. Yields were determined as total cell carbon and ranged from 1.7 to 5.9 g of C mol of S(0) consumed-1 in the presence of the amino acid supplement and from 0.9 to 3.4 g of C mol of S(0-1) in its absence. Several lines of evidence indicate that cell-to-sulfur contact is not required for growth. Approaches for the culture of sulfur-metabolizing bacteria and potential ecological implications of sulfur reduction in Shewanella-like heterotrophs are discussed.     

Mechanism of acetate oxidation to CO2 with elemental sulfur in Desulfuromonas acetoxidans

The strict anaerobe Desulfuromonas acetoxidans can oxidize acetate to CO₂ with elemental sulfur as electron acceptor. ¹⁴C-labelling experiments and enzyme studies are described revealing that acetate oxidation proceeds via the citric acid cycle with the synthesis of oxaloacetate from acetate and 2 CO₂ via pyruvate as anaplerotic reaction. An oxidation of acetate via one carbon unit intermediates as proposed for anaerobic bacteria fermenting acetate to 2 CO₂ and 4 H₂ was excluded.Dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

Anoxic oxidation of arsenite linked to chemolithotrophic denitrification in continuous bioreactors

In this study, the anoxic oxidation of arsenite (As(III)) linked to chemolithotrophic denitrification was shown to be feasible in continuous bioreactors. Biological oxidation of As(III) was stable over prolonged periods of operation ranging up to 3 years in continuous denitrifying bioreactors with granular biofilms. As(III) was removed with a high conversion efficiency (>92%) to arsenate (As(V)) in periods with high volumetric loadings (e.g., 3.5–5.1 mmol As L day^?1). The maximum specific activity of sampled granular sludge from the bioreactors was 0.98 ± 0.04 mmol As(V) formed g^?1 VSS day^?1 when determined at an initial concentration of 0.5 mM As(III). The microbial population adapted to high influent concentrations of As(III) up to 5.2 mM. However, the As(III) oxidation process was severely inhibited when 7.6–8.1 mM As(III) was fed. Activity was restored upon lowering the As(III) concentration to 3.8 mM. Several experimental strategies were utilized to demonstrate a dependence of the nitrate removal on As(III) oxidation as well as a dependence of the As(III) removal on nitrate reduction. The molar stoichiometric ratio of As(V) formed to nitrate removed (corrected for endogenous denitrification) in the bioreactors approximated 2.5, indicating complete denitrification was occurring. As(III) oxidation was also shown to be linked to the complete denitrification of NO to N₂ gas by demonstrating a significantly enhanced production of N₂ beyond the background endogenous production in a batch bioassay spiked with 3.5 mM As(III). The N₂ production also corresponded closely to the expected stoichiometry of 2.5 mol As(III) mol^?1 N₂–N for complete denitrification. Biotechnol. Bioeng. 2010;105: 909–917. © 2009 Wiley Periodicals, Inc.     

硫氧化菌种脱除硫化物生成单质硫限制性因素优化

【目的】针对硫氧化菌种较为特殊的生化特性,优选其氧化硫化物生成单质硫过程的相关限制性因素,以提高该类菌种生成单质硫效率。【方法】采用一株典型脱硫菌Thermithiobacillus tepidarius JNU-2(T.tepidarius JNU-2)氧化硫化物生成单质硫。研究该菌株在以Na2S2O3为能源底物时的培养特性和脱硫性能,并结合单因素实验对菌株氧化硫化物生成单质硫的限制性因素进行优选。【结果】T.tepidarius JNU-2在以Na2S2O3为唯一能源底物培养时的μmax为0.207 h-1,最终生物量为4.0×106 cells/m L。98%的Na2S2O3在24 h时被消耗殆尽,此时单质硫产量达到最大值为0.8 g/L。随后单质硫逐渐被氧化利用,最终稳定在0.2 g/L。经过对该过程主要限制性因素进行单因素实验优化,确定最佳碳氮源、Mg SO4、Fe SO4和能源底物条件分别为:CO2、NH4Cl0.5 g/L、Mg SO4 0.5 g/L、Fe SO4 0.1 g/L和Na2S2O3 15.0 g/L。优化后的氧化Na2S2O3生成单质硫过程的最大生物量可达4.8×106 cells/m L,单质硫产量提升至1.14 g/L。相较于未优化之前,单质硫的产量提高了42.5%。【结论】优化该过程主要限制性因素可有效提高化能自养型T.tepidarius JNU-2氧化硫化物生成单质硫效率。     

Anaerobic oxidation of thiosulfate and elemental sulfur in Thiobacillus denitrificans

Thiobacillus denitrificans strain RT could be grown anaerobically in batch culture on thiosulfate but not on other reduced sulfur compounds like sulfide, elemental sulfur, thiocyanate, polythionates or sulfite. During growth on thiosulfate the assimilated cell sulfur was derived totally from the outer or sulfane sulfur. Thiosulfate oxidation started with a rhodanese type cleavage between sulfane and sulfone sulfur leading to elemental sulfur and sulfite. As long as thiosulfate was present elemental sulfur was transiently accumulated within the cells in a form that could be shown to be more reactive than elemental sulfur present in a hydrophilic sulfur sol, however, less reactive than sulfane sulfur of polythionates or organic and inorganic polysulfides. When thiosulfate had been completely consumed, intracellular elemental sulfur was rapidly oxidized to sulfate with a specific rate of 45 natom S°/min·mg protein. Extracellularly offered elemental sulfur was not oxidized under anaerobic conditions.     

好氧反硝化细菌及其在微污染水源水修复中的应用研究进展

相比于氨氮,天然水体中的硝酸盐氮通常更稳定,导致更难将其从水中去除。由于好氧反硝化可以在有氧环境下进行反硝化作用去除硝酸盐氮,该过程对含有较高溶解氧的天然水体中硝酸盐氮处理有重要作用。本文综述了好氧反硝化菌的分离纯化现状、微生物代谢机制和环境影响因子,并介绍了功能菌群在微污染饮用水源水生物修复的应用研究进展。与一般的厌氧反硝化类似,好氧反硝化菌的种属分布较广,常见的如假单胞菌属(Pseudomoas)、产碱杆菌属(Alcaligenes)、副球菌属(Paracoccus)和芽孢杆菌属(Bacillus)等所属部分微生物均有好氧反硝化能力。大部分好氧反硝化菌株在最佳生长条件下(25–37℃、溶解氧浓度为3–5mg/L、pH为7–8、碳氮比为5–10)具有高效的脱氮效率。但目前好氧反硝化作用在微污染饮用水源水的生物修复方面的应用仍有着脱氮性能不稳定、菌剂流失等不足。此外,目前较少相关中试及实际工程应用的研究,需要进一步的深入探究。     

Growth of the facultative anaerobe Shewanella putrefaciens by elemental sulfur reduction.

The growth of bacteria by dissimilatory elemental sulfur reduction is generally associated with obligate anaerobes and thermophiles in particular. Here we describe the sulfur-dependent growth of the facultatively anaerobic mesophile Shewanella putrefaciens. Six of nine representative S. putrefaciens isolates from a variety of environments proved able to grow by sulfur reduction, and strain MR-1 was chosen for further study. Growth was monitored in a minimal medium (usually with 0.05% Casamino Acids added as a growth stimulant) containing 30 mM lactate and limiting concentrations of elemental sulfur. When mechanisms were provided for the removal of the metabolic end product, H2S, measurable growth was obtained at sulfur concentrations of from 2 to 30 mM. Initial doubling times were ca. 1.5 h and substrate independent over the range of sulfur concentrations tested. In the cultures with the highest sulfur concentrations, cell numbers increased by greater than 400-fold after 48 h, reaching a maximum density of 6.8 x 10(8) cells ml-1. Yields were determined as total cell carbon and ranged from 1.7 to 5.9 g of C mol of S(0) consumed-1 in the presence of the amino acid supplement and from 0.9 to 3.4 g of C mol of S(0-1) in its absence. Several lines of evidence indicate that cell-to-sulfur contact is not required for growth. Approaches for the culture of sulfur-metabolizing bacteria and potential ecological implications of sulfur reduction in Shewanella-like heterotrophs are discussed.     

Molecular analysis of a perchlorate reductase from a perchlorate-respiring bacterium Perc1ace

Perchlorate (ClO₄^–) is a major ground water pollutant of public health concern. ClO₄^– reductase is the key enzyme in the pathway of ClO₄^– breakdown. ClO₄^– reductase from cell-free extracts of the ClO₄^–-respiring bacterium perc1ace was purified 10-fold by ion-exchange and molecular exclusion fast protein liquid chromatography (FPLC). The ClO₄^– reductase catalyzed the reduction of ClO₄^– at a V_max and K_m of 4.8 U mg protein^–1 and 34.5 M, respectively. ClO₄^– reduction was achieved in the temperature range of 20 to 40C and with optimum activity at 25C to 30C and pH 7.5 to 8.0. Molecular masses of two subunits of ClO₄^– reductase were determined by SDS-PAGE to be 35 kDa and 75 kDa. MALDI-TOF/MS analysis of a trypsin digest of the 35 kDa subunit, revealed several tryptic peptides. Amino acid sequences of 22 tryptic peptides of the 35 kDa ClO₄^– reductase subunit were obtained by electrospray mass spectrometry. GenBank protein Blast analysis of the amino acid sequences revealed relevant similarity to reductases, dehydrogenases and heme proteins. Data obtained are useful towards the identification of the overall genetic determinants of ClO₄^– reduction and specific in situ detection of ClO₄^– as well as NO₃-reducing bacteria in ground water.     

Combined removal of sulfur compounds and nitrate by autotrophic denitrification in bioaugmented activated sludge system

An autotrophic denitrification process using reduced sulfur compounds (thiosulfate and sulfide) as electron donor in an activated sludge system is proposed as an efficient and cost effective alternative to conventional heterotrophic denitrification for inorganic (or with low C/N ratio) wastewaters and for simultaneous removal of sulfide or thiosulfate and nitrate. A suspended culture of sulfur-utilizing denitrifying bacteria was fast and efficiently established by bio-augmentation of activated sludge with Thiobacillus denitrificans. The stoichiometry of the process and the key factors, i.e. N/S ratio, that enable combined sulfide and nitrogen removal, were determined. An optimum N/S ratio of 1 (100% nitrate removal without nitrite formation and low thiosulfate concentrations in the effluent) has been obtained during reactor operation with thiosulfate at a nitrate loading rate (NLR) of 17.18 mmol N L(-1) d(-1). Complete nitrate and sulfide removal was achieved during reactor operation with sulfide at a NLR of 7.96 mmol N L(-1) d(-1) and at N/S ratio between 0.8 and 0.9, with oxidation of sulfide to sulfate. Complete nitrate removal while working at nitrate limiting conditions could be achieved by sulfide oxidation with low amounts of oxygen present in the influent, which kept the sulfide concentration below inhibitory levels.     

The production and utilization of intermediary elemental sulfur during the oxidation of reduced sulfur compounds by Thiobacillus ferrooxidans

The intermediary production of elemental sulfur during the microbial oxidation of reduced sulfur compounds has frequently been reported. Thiobacillus ferrooxidans, an acidophilic chemolithoautotroph, was found to produce an insoluble sulfur compound, primarily elemental sulfur, during the oxidation of thiosulfate, trithionate, tetrathionate and sulfide. This was confirmed by light and electron microscopy. Sulfur was produced from sulfide by an oxidative step, while the production from tetrathionate was initiated by a hydrolytic step, probably followed by a series of chemical reactions. The oxidation of intermediary sulfur was severely inhibited by sulfhydryl-binding reagents such as N-ethylmaleimide, by the addition of uncouplers or after freezing and thawing of the cells, which probably damaged the cell membrane. The mechanisms behind these inhibitions have not yet been clarified. Finally, it was observed that elemental sulfur oxidation by whole cells depended on the medium composition. The absence of sulfate or selenate reduced the sulfur oxidation rate.Non-standard abbreviations NEM N-ethylmaleimide - CCCP carbonyl cyanide m-chlorophenyl hydrazone